Apparatus and method for digital holographic imaging

ABSTRACT

The present invention is related to a method for performing digital holographic imaging (DHI), said DHI being characterized by the fact that images of a sample are obtained by applying numerical means to reconstruct holograms of the sample. In the method of the invention, the sample is in a medium with controlled properties that influence the behaviour of the sample, and/or that influence the process of hologram formation. The information content of one hologram, or of a plurality of holograms, recorded with the sample in one medium, or in a plurality of medium, is used to reconstruct one image of the sample, or a plurality of images of the sample, that describe quantitatively one property of the sample, or a plurality of properties of the sample. The present invention is also related to an apparatus with which to perform said method.

FIELD OF THE INVENTION

The invention relates to digital holographic imaging (DHI) apparatusesand methods which provide an holographic representation of a sample.

STATE OF THE ART

Holography is a three-dimensional (3D) imaging technique that makes useof the interference between a reference wave and a wave emanating fromthe sample called object wave. The purpose of this interference is torecord the phase of the object wave, which is related to the 3Dcharacter of the sample. With digital holographic imaging (DHI),real-time observations can be achieved by using a charged coupled device(CCD) camera as recording device and by performing a numericalreconstruction of the hologram. This idea has been proposed for thefirst time over 30 years ago by J. W. Goodmann, R. W. Lawrence, in“Digital image formation from electronically detected holograms,” Appl.Phys. Lett, Vol. 11, 1967. As a result of technological progressesachieved in the fields of digital image acquisition and processing, thisnumerical or digital approach of holography has considerably extendedthe fields of its potential applications and different types ofDHI-inspired imaging systems have been developed during the last years.

DHI techniques can be classified in two main categories: in-linetechniques characterized by the fact that the reference and object waveshave similar propagation directions, and off-axis techniques for whichthe two interfering waves propagates along different direction. Theprocedure for hologram formation in in-line digital holography issimilar to the procedure used for phase measurements with so-calledphase-shifting interferometric techniques. Hologram formation within-line techniques requires the acquisition of several images, at leastthree, that must be recorded during a modulation of the reference phase.Off-axis techniques, are more simple from the experimental point of viewsince they require a single hologram acquisition without modulation ofthe phase of the reference wave. In-line techniques however present theadvantage that the reconstructed images are free of twin images and zeroorder of diffraction. Among off-axis techniques, we can distinguishmethods based on Fourier-transform holography, and methods based on aso-called Fresnel holography. With Fourier-transform methods thereference wave must be a spherical wave of precisely controlledcurvature and image reconstruction is basically performed by Fouriertransformation of the hologram. With Fresnel-holography basedtechniques, the reconstruction procedure is more sophisticated but moreflexibility is offered to build experimental installations.

Among recent publications presenting developments or applications ofDHI-inspired techniques, we can mention the following works. A study ofsome general performances of an in-line technique is presented in “Imageformation in phase-shifting digital holography and application tomicroscopy”, I. Yamaguchi et al., Applied Optics, Vol. 40, No. 34, 2001,pp. 6177-6186. In “Fourier-transform holographic microscope,” AppliedOptics, Vol. 31, 1992, pp. 4973-4978, W. S. Haddad et al describe thegeneral principle of Fourier-transform DHI. Examples of applications ofthe Fresnel-based approach can be found in “Direct recording ofholograms by a CCD target and numerical reconstruction,” U. Schnars andW. Jüptner, Applied Optics, Vol. 33, 1994, pp. 179-181, and in“Performances of endoscopic holography with a multicore optical fiber,”O. Coquoz et al., Applied Optics, Vol. 34, 1995, pp. 7186-7193.

A key element of a DHI method is the numerical method used for hologramreconstruction. An original reconstruction procedure, which allows forreconstructing simultaneously the amplitude and the phase of the objectwave, on the basis of a single off-axis hologram acquisition, has beendeveloped by Cuche et al and is presented in U.S. Pat. No. 6,262,218,and in WO 00/20929. Different applications and implementations of thistechnique are presented in “Digital holography for quantitativephase-contrast imaging”, Optics Letters, Vol. 24, 1999, pp. 291-293, in“Simultaneous amplitude-contrast and quantitative phase-contrastmicroscopy by numerical reconstruction of Fresnel off-axis holograms”,Applied Optics, Vol. 38, 1999, pp. 6994-7001, in “Spatial Filtering forZero-Order and Twin-Image Elimination in Digital Off-Axis Holography”,Applied Optics, Vol. 38 No. 34, 1999, in “Aperture apodization usingcubic spline interpolation: Application in digital holographicmicroscopy”, Optics Communications, Vol. 182, 2000, pp. 59-69, and in“Polarization Imaging by Use of Digital Holography”, T. Colomb et al.,Applied Optics, Vol. 38, No 34, 1999.

DHI method presents interesting possibilities of applications in cellbiology. Indeed a living cell behaves optically as a phase object, i.e.a transparent sample whose constituents can be optically probed on thebasis of the phase shift they induce on the light crossing them. Thephase-shifting behavior of transparent sample is well known, and for along time as it constitutes the mechanism of image formation inphase-contrast (PhC) and Nomarski (DIC) microscopy. Even though thesetwo techniques are widely used in biological microscopy, and well suitedas contrasting methods, they cannot be used for precise quantitativephase measurements. The DHI method instead, is reminiscent of classicalinterferometry, which is the most commonly used technique for phasemeasurements. However, whereas interferometric techniques are widelyused in metrology, only few biological applications have been reported,by R. Barer and S. Joseph, in “Refractometry of living cells”, QuarterlyJournal of Microscopical Science, Vol. 95, 1954, pp. 399-423, by .R.Barer in “Refractometry and interferometry of living cells,” Journal ofthe Optical Society of America, Vol. 47, 1957, pp. 545-556, by A. J.Coble et al. in “Microscope interferometry of necturus galibladerepithelium”, Josiah Macy Jr. Fundation, New York, 1982, p. 270-303, byK. C. Svoboda et al in “Direct observation of kinesin stepping byoptical trapping interferometry”, Nature, Vol. 365, 1993, by J. Farinasand A. S. Verkman, in “Cell volume plasma membrane osmotic waterpermeability in epithelial cell layers measured by interferometry,”Biophysical Journal, Vol. 71, 1996, pp. 3511-3522, by G. A. Dunn and D.Zicha in “Dynamics of fibroblast spreading,” Journal of Cell Science,Vol. 108, 1995, pp. 1239-1249.

For biological applications, as well as for material science ormetrology applications, DHI methods offer a novel alternative toclassical interferometry with similar performances but simplifiedexperimental procedures. The main advantage originates from the factthat complex and costly experimental optical devices can be handled bydigital processing methods. For example, as described by E. Cuche et al.in “Simultaneous amplitude-contrast and quantitative phase-contrastmicroscopy by numerical reconstruction of Fresnel off-axis holograms”,Applied Optics, Vol. 38, 1999, pp. 6994-7001, the wave frontdeformations appearing when a microscope objective is introduced alongthe path of the object wave can be compensated using a digitalprocedure. This particular feature opens attractive possibilities in thefields of microscopy. In addition DHI techniques performs faster thaninterferomeric techniques, and provides more information about thesample, in particular, the amplitude and the phase of the object wavecan be obtained simultaneously on the basis of a single hologramacquisition.

DHI methods have been applied to static imaging of biological cells,without phase reconstruction by K. Boyer et al. in “Biomedicalthree-dimensional holographic microimaging at visible, ultraviolet andX-ray wavelength”, Nature Medecine, Vol. 2, 1996, pp. 939-941, and by F.Dubois et al. in “Improved three-dimensional imaging with a digitalholography microscope with a source of partial spatial coherence,”Applied Optics, Vol. 38, 1999, pp. 7085-7094. DHI of cells using a phasemeasurement modality requiring several image acquisitions has beenreported by G. Indebetouw and P. Klysubun in “Saptiotemporal digitalmicroholography,” Journal of the Optical Society of America A, Vol. 18,2001, pp. 319-325.

With DHI, image acquisition can be performed at video-rate, and evenfaster using appropriate image acquisition systems, for experimentalperiods of up to several hours. Due to its interferometric nature, DHIhas a high axial resolution (nanometer scale), which allows forobserving subtle and minute modifications of sample shape, opening awide field of applications in both life and material sciences.

A well-known limitation of interferometric techniques in general, and ofDHI methods in particular, comes from the fact that the measuredquantity, i.e. the phase of an electromagnetic radiation, can besimultaneously influenced by several factors. When an electromagneticradiation interact with a sample, the result of this interaction, and inparticular the changes occurring on the phase of the incident radiation,depends on three main classes of parameters:

a) Geometrical properties of the sample. The surface topography for areflective sample imaged in reflection and the thickness of the samplefor a transparent sample imaged in transmission.

b) Dielectric properties of the sample. The absorption that influencesthe intensity of the radiation, and the index of refraction thatmodifies the phase of the radiation. Absorption and index of refractionare often combined in the concept of complex index of refraction.

c) Dielectric properties of the medium incorporating the sample. It isonly in the vacuum, or by approximation in the air, that anelectromagnetic interaction depends only on the properties of thesample. In the general case, the dielectric properties of a sample mustbe defined relatively to the dielectric properties of the mediumincorporating the sample. For example, its is not the absolute index ofrefraction of the sample (n_(s)) that must be considered, but thedifference (n_(s)−n_(m)), where n_(m) is the index of refraction of themedium incorporating the sample.

It is therefore often difficult to interpret data resulting from themeasurement of the phase of an electromagnetic radiation. For example,if one wants to determine precisely and unambiguously the shape of asample, the knowledge of the dielectric properties (of both the sampleand the medium) is required. And if one wants to determine thedielectric properties of a sample, the shape of the sample and thedielectric properties of the medium must be known. A rigorous andunambiguous interpretation of the results can be obtained for example bytaking dielectric property values from the literature, or by measuringthe geometry or the dielectric properties of the sample with anothertechnique.

OBJECTIVES OF THE INVENTION

The present invention aims at providing an apparatus and a method forperforming digital holographic imaging (DHI) which consider the mediumincorporating the sample as an active parameter of the image formationprocess. It is also an objective that such an apparatus and methodimprove the performances of DHI by providing more accurate measurements.It is another objective to study dynamic effects resulting frominteractions between the sample and the incorporating medium. Finally,another objective is to provide more information about the sample, inparticular concerning its geometrical and dielectric properties.

SUMMARY OF THE INVENTION

The present invention concerns an apparatus as defined in claim 1, amedium as defined in claim 6 and a method for performing digitalholographic imaging (DHI) of a sample as defined in claim 7.

Some embodiments of the invention which relate to an apparatus aredefined in subclaims 2 to 5. Other embodiments which relate to a methodare defined in subclaim 8 to 13.

The invention is particularly useful with a method or a device asdisclosed in PCT patent application WO 00/20929 and in particular with adevice comprising an hologram creation device which includes a source ofradiation, means to illuminate the sample unit with said radiation,means to create an object wave by collecting the radiation afterinteraction with the sample unit, means to create a reference wave fromsaid radiation, means to produce a hologram by interference between saidreference wave and said object wave, means to produce a digital hologramby acquiring and digitizing said hologram, arid means to transmit saiddigital hologram to said hologram reconstruction unit, and wherein saidhologram reconstruction unit provides an amplitude-contrast image and/ora quantitative phase contrast image by numerical calculations applied tosaid digital hologram, and wherein said processing unit determine thedielectric properties of the sample and/or the shape of the sample byprocessing a plurality of said amplitude contrast images and/or aplurality of said quantitative phase-contrast images, and wherein saidprocessing unit can also be used to describe the evolution of saidsample by processing a plurality of said amplitude contrast imagesand/or a plurality of said quantitative phase-contrast images.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the building block of an apparatusaccording to the present invention.

FIG. 2 refractive index (n) and absorption coefficient (k) behaviorsnear an absorption band.

FIG. 3 presents an example of application of the method of the presentinvention. FIG. 3 a schematically represent a biological cell in aperfusion chamber. FIG. 3 b shows an example of a hologram of abiological cell. FIG. 3 c shows an example of a quantitativephase-contrast image of a biological cell. FIG. 3 d shows an example ofa quantitative phase-contrast image of a biological cell presented in 3Dperspective.

FIG. 4 presents an example of application of the method of the presentinvention. FIG. 4 a shows an example of the evolution during time of thephase measured in a specific area of a cell. FIG. 4 b shows an exampleof a quantitative phase-contrast image of a cell. FIG. 4 c shows anexample of a concentration-response curve established thanks to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

An important aspect of the invention is represented by the medium whichincludes the sample. As schematically shown in FIG. 1, this means thatthe sample 1 is located inside a given volume, defined for example by acontainer 2, filled with a medium 3. The sample unit 4 is composed ofthe sample 1, the container 2 and the medium 3. The medium 3 can be agas, a liquid, a gel, a solid, a powder or a mixture of solid particlesin suspension in a liquid. If the medium 3 is a gas, the container 2 isclosed. If medium 3 is a liquid, the container 2 can also be closed, butthe top face of the container 2 can be opened. If medium 3 is solid or agel, container 2 is optional. For example, the sample 1 can be a livingsample embedded in a physiological solution.

In particular, medium 3 may be composed of, or may comprise, elementsthat change the index of refraction of medium 3. For example, medium 3can be a liquid element comprising different concentrations of moleculessuch as metrizamide (C₁₈H₂₂I₃N₃O₈), and/or different concentrations ofmanitol, and/or iohexol, and/or iodixanol, and/or ficoll, and/orpercoll.

In particular, medium 3 may be composed of, or may comprise, elementsthat have dispersion properties, meaning that the dielectric propertiesof medium 3 are different for different wavelength of theelectromagnetic spectrum. For example, medium 3 may compriseconcentrations of an organic molecule, and/or of an inorganic molecule,and/or of a dye, and/or of a stain, and/or of a chromophore, and/or ofpigments, and/or of a colloid, and/or of a neutral stain, and/or of adiazotic compound, and/or of a triphenylmethan compound, and/or of axanthen compound, and/or of a aminoxanthen compound, and/or of ahydroxyxanthen compound, and/or of pyronyn, and/or of rhodamin, and/orof fluorescein, and/or of eosin, and/or of carbocyanin, and/or ofoxadicarbocyanin, and/or of methylen blue, and/or of phthalocyanin,and/or of a ph-indicator element, and/or of sulforhodamin, and/or oftrypan blue, and/or of thiazin, and/or of fast green FCF, and/or ofevan's blue.

In particular, medium 3 may be a mixture of particles, micro- and/ornano-particles in particular, in suspension in a liquid. For example,these particles may be metallic particles, and/or dielectric particles,and/or coated particles, and/or gold particles, and/or silver particles,and/or copper particles, and/or silicon particles, and/or crystallineparticles, and/or semi-conductors particles, and/or CdS particles,and/or CdSe particles, and/or latex beds, and/or charged particles,and/or colloidal gold particles, and/or silver particles.

A medium controller device 5 can be optionally used, more particularlyif medium 3 is a gas or a liquid. The role of the medium controllerdevice 5 is to permit exchanges of medium 3. When medium 3 is composedof a plurality of constituents, the medium controller device 5 can beused to change the concentration of one of these constituents, or of aplurality of these constituents. The medium controller device 5 can alsocomprise means to control the temperature, and/or the pressure of medium3. It may also include means to control gas partial pressures such asoxygen or carbon dioxide. The medium controller device 5 can alsocomprise means, to induce electrical or magnetic stimulations of thesample 1 or of the entire sample unit 4. In particular, electrodes orelectrode arrays may be included in the medium control device 5. Themedium controller device 5 can also comprise means to induce amechanical stimulation of the sample, for example a tip and a cantileveras performed in atomic force microscopy (AFM).

In particular, medium 3 may be a mixture of particles, micro- and/ornano-particles in particular, in suspension in a liquid, and the mediumcontroller device 5 may be used to control the type of these particles,and/or the concentration of these particles, and/or the sizes of theseparticles.

When introduced in a set-up for digital holographic imaging (DHI), thesample unit 4 is illuminated by an incident radiation calledillumination wave 6. After interaction with the sample unit, theincident radiation becomes the object wave 7. The incident radiation isprovided by a radiation source 8, which emits preferentially radiationsof the electromagnetic type. It is clear that to enable interactionbetween the sample 1 and the illumination wave 6, at least one face ofcontainer 2 comprises a zone that is transparent for the incidentradiation.

The medium controller device 5 may comprise means, such aspiezo-electric or acoustic transducers, to induce mechanical vibrationsof the sample unit 4 or a vibration of medium 3. Such vibrations areexpected to have a positive impact on the signal to noise ratio (SNR) ofthe method by inducing a random variation of the propagation directionsof parasitic radiations emitted by elements of the container 2 thatinteract with the illumination wave 6.

The standard procedure for digital holographic imaging (DHI) isdescribed for example in patent WO 00/20929. We just give here a summaryof the most important steps of the DHI process. The object wave 7 iscollected using appropriate means 9 (e.g. a lens, an assembly of lensesor a microscope objective), and appropriate means (e.g. an assembly beamsplitters, mirrors, lenses and apertures) are used to produce areference wave 10 from the radiation source 8. Appropriate means 11(e.g. an assembly beam splitters, mirrors, lenses and apertures) areused to form a hologram 12 by making the reference wave 10, and theobject wave 7., to interfere with each other. The hologram 12 isacquired by an image acquisition system 13, which transmit a digitalhologram 14 to a hologram reconstruction unit 15. The reconstructionunit 15 applies numerical calculations to reconstruct an amplitudecontrast image 16 and/or a quantitative phase contrast image 17. Anadditional processing unit 18 can be used to collect and to process aplurality of said amplitude contrast images and/or a plurality of saidquantitative phase contrast images. The main function of the processingunit 18 is to provide results that are specific to the method of thepresent invention. In particular, the processing unit 18 can be used todescribe the evolution of the sample 1 during time, and/or to providemeasurements and a representation its dielectric properties, and/or toprovide measurements and a representation of its thickness, and/or toprovide measurements and a representation of its topography.

The processing unit 18 can also be used to improve the accuracy of themeasurements by applying statistical procedure to a plurality of saidamplitude contrast images 16 and/or to a plurality of said quantitativephase contrast images 17.

When a plurality of amplitude contrast images 16 and/or a plurality ofquantitative phase contrast images 17 are provided during a given periodof time, the processing unit 18 can be used to process temporal signalsextracted from the plurality of amplitude contrast images 16 and/or theplurality of quantitative phase contrast images. For example, theprocessing unit can be used to Fourier-transform said temporal signals,and/or can be used to apply band-pass, and/or low-pass, and/or high passfiltering techniques to said temporal signal.

Alternatively a plurality of amplitude contrast images 16 and/or theplurality of quantitative phase contrast images 17 can be collectedafter successive modifications of the irradiation wavelength λ. If theholograms are recorded in the transmission geometry with a semitransparent sample, and provided that the dispersion law of therefractive index is known, a tomographic image of the sample can beobtained

In another alternative a plurality of amplitude contrast images 16and/or the plurality of quantitative phase contrast images 17 can becollected at variable incidence angles of the irradiating beam. Thisachievement can be obtained by rotating the sample. In anotherembodiment, the sample can be kept fixed and the irradiating beam can bemoved in order to vary the incidence angle of the irradiating beam overthe sample. The processing unit 18 can be used to operate a Radontransform of the collected data. Furthermore, such a mean can beoperated to compute the local dielectric properties of the sample byapplying an algorithm of back-projection. Fourier slice theorem inparticular can be applied to the calculation of the dielectric potentialof the sample.

In another alternative a plurality of amplitude contrast images 16and/or the plurality of quantitative phase contrast images 17 can becollected after successive modifications of the irradiation wavelength λand after successive modifications of the incidence angles of theirradiating beam. The processing unit 18 can be used to compute thelocal dielectric properties of the sample by applying an algorithm.Fourier slice theorem as commonly applied in diffraction tomography.

The processing unit 18 can also be used to generate representations ofthe obtained results, for example by producing three-dimensionalperspective representations of the obtained images, and/or by generatingso-called false colors representations of the obtained images, and/or bygenerating movies or animations from a plurality of obtained images.

When a plurality of images are provided, the processing control unit canalso be used to compare them which each other, for example in order toreveal the spatial distribution of signal changes.

The most important physical parameters describing a typical applicationof the present invention are:

-   -   φ is the phase of the object wave 7, expressed in degree or in        gradient. φ is provided by DHI as an image called quantitative        phase-contrast image 17.    -   OPL=φλ/2π, where λ is the wavelength of the radiation. OPL is        called optical path length. When φ is measured in degree, we        have OPL=φλ/360.    -   n_(m) is the index of refraction, also called refractive index,        of the medium incorporating the sample, or more precisely the        index of refraction of medium 3.    -   n_(s) the index of refraction of the sample 1.    -   d the thickness of the sample 1.    -   h the height of the sample 1, which describes the topography of        the sample.        All parameters described in the list above are provided in the        form two-dimensional (2D) functions describing the spatial        distribution of these parameters in a specific plane, or in        other words in the form of images. It is clear when these images        are provided in a digital form, the corresponding parameter can        be measured from the values of the pixels composing the digital        images.

In a transmission imaging geometry, which is characterized by the factthat the object wave 7 is collected after the illumination wave 6 hascrossed the sample unit 7, the quantity measured in one pixel of saidquantitative phase-contrast image maps, the 2D distribution of the phaseshifts induced by the sample 1 on the radiation crossing the sample 1.The corresponding OPL can be expressed as follows:OPL=(n _(s) −n _(m))d  (1),Expression (1) clearly indicates that the measured signal depends on thestructural properties of the sample (its thickness d), and on thedifference between the dielectric properties of the sample, and thedielectric properties of the medium incorporating the sample(n_(s)−n_(m) the difference of index of refraction).

In a reflection imaging geometry, characterized by the fact that theobject wave 7 is collected after the illumination wave 6 has beenreflected or backscattered by the sample, and if we consider only samplehaving surfaces with constant and homogeneous dielectric properties, thequantity measured in one pixel of said quantitative phase-contrast imagedepends on the surface topography of the sample and on the index ofrefraction of the medium incorporating the sample n_(m). Thecorresponding OPL can be expressed as follows:OPL=2n _(m) d  (2),

Expressions (1) and (2) gives only examples of expressions describingthe behavior of OPL as a function of the specimen shape and dielectricproperties. The present invention can also be applied determine thespecimen shape and the dielectric properties of the specimen on thebases of more complex or more general models describing the interactionof an electromagnetic radiation with a specimen. In particular, suchmodels can be obtained within the framework of the Maxwell equations,and/or within the framework of a vectorial or tensorial theory ofelectromagnetic interaction, and/or within the framework of thescattering potential equation, and/or within the framework oftheoretical model describing the diffraction of an electromagneticradiation by a specimen. It is also clear that such models may involveother dielectric properties than the refractive index, in particular theabsorption, and/or the complex index of refraction. In particular suchmodels may give more accurate results for small samples, and/or when thedielectric properties of the sample and the dielectric properties ofmedium are significantly different.

It is an object of the present invention that the proposed method andapparatus can be used to study the effect of a medium on the sample. Inthis case, the medium controller device 5 can be used to control thequantity of a given medium in the medium incorporating the sample. Ifthis medium interact with the sample and induces OPL variations, thesesOPL variations can be measured and monitored during time with DHI.

It is an object of the present invention that the medium incorporatingthe sample can be used to modulate the OPL signal measured by DHI. Moreparticularly, and as clearly stated by expressions (1) and (2), changingn_(m) has a direct influence on the measured OPL signal. In particular,for small or thin sample producing weak OPL signals, an OPL signalamplification can be achieved by increasing n_(m) in reflection, or byincreasing n_(s)−n_(m) in transmission.

Changing n_(m) the index of refraction of the medium incorporating thesample can be achieved by changing medium 3 using the medium controllerdevice 5. In particular, the medium controller device 5 can be used tocontrol the composition of medium 3, said composition comprisingelements that changes the index of refraction of the medium. Forexample, medium 3 can be a liquid element comprising differentconcentrations of molecules or ions. For example medium 3 can compriseelements such as metrizamide (C₁₈H₂₂I₃N₃O₈), and/or manitol, and/oriohexol, and/or iodixanol, and/or ficoll, and/or percoll. For example,medium 3 can be a mixture of particles in suspension in a liquid, andthe index of refraction of the medium 3 can be changed by changing thedistribution of the sizes of these particles, and/or by changing thestatistical distribution of the size of these particles, and/or bychanging the concentration of these particles, and/or by changing thematerial of these particles.

It is an object of the present invention that the medium 3 envelopingthe sample 1 may be composed of, or may comprise, an element havingdispersion properties, meaning that the index of refraction of themedium incorporating the sample n_(m) is different for differentwavelength of the radiation. Therefore, n_(m) can be changed by changingthe wavelength λ of the radiation illuminating the sample unit 4.

For example the medium having dispersion properties can be characterizedby an absorption spectrum showing absorption lines or absorption bands.According to the Kramers-Kronig relationship the dispersion behavior ofsuch a medium, in a wavelength interval near an absorption line or nearan absorption band, is characterized by the fact that importantrefractive index changes can occur even in small wavelength intervals.For example, as described in FIG. 2, two wavelengths λ₁ and λ₂, can bechosen so that they are located along the decreasing side of anabsorption band, where dispersion effects are more pronounced. Thisenables fast and important modulations of n_(m).

Particularly in the transmission imaging geometry, when a plurality ofquantitative phase-contrast images have been obtained for differentvalues of n_(m), the thickness of the sample (d), and the index ofrefraction of the sample n_(s), can be obtained separately orsimultaneously, assuming that the different values of n_(m) are known orhave been measured previously. For example, lets define OPL1 and OPL2,two different OPL measurements performed for two different values ofn_(m), respectively n_(m1) and n_(m2). From expression (1), we have:OPL 1=(n_(s) −n _(m1))d  (3),OPL 2=(n_(s) −n _(m2))d  (4),and n_(s) and d can be directly determined as follows:d=(OPL 1−OPL 2)/(n _(m2) −n _(m1))  (5),n _(s)=(OPL 2 n _(m1) −OPL 1 n _(m2))/(OPL 2−OPL1)  (6).Even if two image acquisitions are sufficient, more acquisitions can beperformed for different values of n_(m), or for the same value of n_(m),for example in order to increase the accuracy of the measurements.

Different values of n_(m) (for example the two values n_(m1) and n_(m2))can be obtained by changing the medium. Different values of n_(m) (forexample the two values n_(m1) and n_(m2)) can also be obtained by usinga medium having dispersion properties that allows to change its index ofrefraction by changing the wavelength of the radiation illuminating thespecimen unit.

The invention can be used to measure n_(m). In the transmission imaginggeometry for example, if d and n_(s), are known, n_(m) can be determinedas follows: n_(m)=n_(s)−OPL/d. More generally, the present invention canbe used as a technique for refractometry when the geometry of thespecimen is known.

The invention can also be used to obtain a tomography of the sample.Provided that n_(m), can be chosen reasonably close to n_(s) in order toavoid significant refraction effects, and provided that n_(m), and n_(s)don not vary to much over a distance comparable to the wavelength λ, inorder to avoid significant diffraction effects, we have in a firstapproximation for a 2D geometry: $\begin{matrix}{{{OPL}\left( {\theta,r} \right)} = {\underset{\underset{unit}{Specimen}}{\int\int}{\mathbb{d}x}{\mathbb{d}y}\quad{n\left( {x,y} \right)}{\delta\left( {{x\quad\cos\quad\theta} + {y\quad\sin\quad\theta} - r} \right)}}} & (7)\end{matrix}$where n(x,y) is the local refractive index which is equal to n_(m)outside the sample and approximately n_(s) with local variations overthe volume of the sample. δ is the Dirac delta distribution.OPL(θ,R)data can be obtained by collecting a plurality of amplitudecontrast images 16 and/or a plurality of quantitative phase contrastimages 17 can be collected at variable incidence angles θ of theirradiating beam. From the collected data the refractive index n(x,y) ofthe sample can be computed by applying an algorithm of back-projection.

If medium 3 may have no influences on the sample, the invention can beused to measure a property or a behavior of the medium 3. For example,if medium 3 is a liquid, the present invention can be used to study, orto observe, dynamics fluid phenomenon introducing local variations ofOPL, flow analysis in particular, for example with flows tracers. Forexample, and more particularly when medium 3 is a solid or a gel, thepresent invention can also be used to analyze internal stresses insidethe material.

As described by T. Colomb et al. in “Polarization Imaging by Use ofDigital Holography”, Applied Optics, Vol. 38, No 34, 1999, DHI can beused to image and to measure the polarization sate of the object wave.It is an object of the present invention that, in addition toamplitude-contrast images 16, and quantitative phase-contrast images 17,the polarization sate of the object wave, may be provided to theprocessing unit 18. In this case, the present invention can also be usedto observe and to analyses processes that influence the polarization ofthe radiation, and in particular dynamic processes inducing transientmodifications of the polarization. In this case for example, the medium3 may have birefringent properties.

If a plurality of holograms are acquired during a period of time, thecorresponding plurality of quantitative phase contrast images can beprocessed, for example by the processing unit 18, in order to compensatetemporal variation of the OPL or phase signal, said temporal variationshaving other origins than the behavior of the sample, for example suchtemporal variation may be caused by mechanical vibrations of elementscomposing the apparatus used to generate holograms, and/or may be causedby air turbulences and/or may be caused by thermal effects. Tocompensate such temporal variations, the present invention uses acomputer-based method that measures the phase signal in regions of thequantitative phase-contrast images where the sample has no influence onthe OPL signal, or in other words in regions where d, or h, or n_(m)have no influence on the measured signal. In such regions, OPLmeasurements provide a reference signal that monitor the influence ofundesirable sources of signal variations. The compensation of theinfluence of these undesirable sources of signal variations can beperformed by subtracting the reference signal from the signal measuredin other area of the quantitative phase-contrast images, in particularin area where the sample influences the measured signal.

Within the general context of imaging and microscopy techniques, DHImethods in general, and the present invention in particular, appear as apowerful mean for real-time imaging of processes inducing dynamicchanges of a sample shape and/or dynamic changes of physical propertiesof a sample. These dynamic changes may appear in relation to aspontaneous process and/or in relation to an induced process. Forexample, DHI methods can be applied to monitor the response of a sampleto external perturbations of different forms, for example chemicalperturbations, and/or biological perturbations, and/or electricalperturbations, and/or mechanical perturbations.

DHI methods in general, and the present invention in particular, presentattractive possibilities for multifunctional imaging approachescombining DHI with other types of existing imaging techniques, and/orWith other types of measurement techniques. For example, the presentinvention can be combined with fluorescence microscopy, or with laserscanning confocal microscopy, or with two-photon microscopy, or withsecond or third harmonic generation microscopy, or with atomic forcemicroscopy. The present invention can also be applied in parallel withexperimental techniques used in electrophysiology, and/or in fluidmechanics.

DHI techniques in general, and the present invention in particular, areparticularly well adapted for non-invasive analysis, because no contrastagents are required, and because low radiation intensities are used. Inparticular, it has been established that the measured irradiance at thesample plane may be several orders of magnitude lower than theirradiance used for example by other microscopy or imaging techniques.It is therefore an interesting potential application of the presentinvention that the method can be applied to image delicate sample, andbiological sample in particular. For example, the sample can be abiological cell, a living biological cell, a culture of biological cell,a mono-layer of cultured biological cells, a tissue composed ofbiological cell, a biochip, a preparations of proteins.

A particular aspect of the method according to the invention ischaracterized by the fact that the medium incorporating the sample canbe changed during time and/or that properties of the mediumincorporating the sample can be changed during time. Such changes of themedium are expected to induce modifications of the sample shape and/ormodifications of the dielectric properties of the sample, and saidmodifications of the sample are monitored by DHI.

As a result of the high acquisition frequency of the DHI method, themethod of the present invention is particularly well adapted to studythe dynamics of fast sample-medium reactions. As DHI is also anon-invasive imaging technique working without contrast agents and withlow radiation intensities, the method of the present invention isparticularly well adapted to study the dynamic of reactions withminimized external perturbations, and during long time periods of hoursor even days. This is of particular interest for applications in lifesciences.

FIG. 3 presents an example of application of the present invention toimage living cells in culture enclosed in a perfusion chamber 30 filledwith a physiological solution 37. As shown in FIG. 3 a, the cell culture31 is illuminated by an incident radiation 32 and a microscope objective33 collects the transmitted radiation to form an object wave 34. A CCDcamera 35 records the hologram. An example of hologram is presented inFIG. 3 b. The hologram is produced by the interference with a referencewave 36. A numerical procedure is then applied to reconstruct thehologram. An example of quantitative phase-contrast image is presentedin FIG. 3 c. The obtained phase-contrast image can be presented in 3Dperspective, as presented for example in FIG. 4 d.

From the quantitative phase-contrast image, quantitative data about thecell morphology can be obtained. The refractive index, at the wavelengthof 633 nm, of the perfused solution n_(m)=1.3336±0.0002, was measuredwith an Abbe refractometer. By assuming, in first approximation, aconstant and homogeneous cellular refractive index n_(s)=1.365, one canestimate that an phase of 10 degree corresponds to a cellular thicknessd=0.6 μm. Using this relation between phase and thickness, phasemeasurements extracted from specific pixels of the quantitativephase-contrast image presented FIG. 3 c, yields a thickness of 1-3 μmfor the neuronal processes and of approximately 8 μm for the cell body.Even with the assumption of a homogeneous distribution of intracellularrefractive index (which can be discussed given a certain degree ofheterogeneity of its constituents), these estimations of cell morphologygive realistic values of typical cell dimensions as described in theliterature for the mouse cortical neurons presented here. Using the sameassumption for refractive index of the cell, and based on a phasemeasurement accuracy of 0.7 degree, that is dictated by experimentalopto-mechanical noise, we obtain an axial (or vertical) resolution of 40nanometers.

As an example of application of the present invention, the presentinvention has been applied to study the effects of an excitatoryneurotransmitter (glutamate) on the morphology of cultured mousecortical neurons. The influence of glutamate on neuron morphology hasbeen already described as resulting from excitotoxicity, for highconcentrations (20-500 μM) and long exposures of several minutes. Thanksto its unique features, the method of the present invention has madepossible the observation of reversible morphological changes occuringfor short applications (a few seconds), at physiological concentrations(EC5 ₅₀=7.5 μM). It has also been established that this effect occurs inresponse to the activation of N-methyl-D-aspartate (NMDA) receptors.Results of the real-time observation of glutamate-mediated morphologicalchanges in neurons are summarized in FIGS. 4 and 5. FIG. 4 a presentsthe temporal evolution of the phase signal measured, in real-time byDHI, in the domain delimited by a black rectangle in FIG. 4 b, whichpresents a typical example of a quantitative phase contrast image of aliving mouse cortical neuron immersed in a physiological solution. Cellswere observed in a control solution (HEPES-buffered physiologicalsolution) and pharmacological agents were applied as short pulses, of 20seconds duration. These pulses of pharmacological agents are indicatedby black triangles in FIG. 4 a. Marked phase increases occur whenglutamate (GLUT) is applied alone, at instants indicated by blacktriangles 41, 42, 43 and 44. The concentrations of GLUT, arerespectively 15 μM, 10 μM, 15 μM and 20 μM. At instants indicated byblack triangles 45 and 46, 400 μM of kynurenate (KYN), a broad spectrumionotropic glutamate receptor antagonist, has been added to GLUT. Noincrease of the phase signal occurs in this case, indicating that theobserved effect of GLUT is inhibited by KYN. The effect of GLUT is alsoinhibited when 5 μM of MK-801, an NMDA specific receptor antagonist, hasbeen added to GLUT in the physiological solution, at instants indicatedby black triangles 47 and 48. As can be seen in FIG. 4 a, theapplication of glutamate pulses induced reversible OPL or phaseincreases lasting for several minutes. These OPL increases could bereproduced several times over periods of more than three hours. FIG. 4 ashows also that these glutamate-mediated responses were inhibited bykynurenate, a broad-spectrum ionotropic glutamate receptor antagonist,and by MK-801, a specific NMDA receptor antagonist, thus stressing thereceptor specific nature of the effect. A concentration-response curve(FIG. 4 c) has been established on the basis of 5 different experiments.As response amplitudes vary from cell to cell, results were normalizedwith respect to the maximal response which occurred at a concentrationof ˜15 μM. Curve fitting using the Hill equation indicates a highlycooperative behavior (Hill coefficient=3.75±0.4) with an EC₅₀ of˜7.5±0.23 μM. Interestingly, these results have been obtained in thepresence of glycine (3 μM) in the perfusion solutions. Without glycinecells started to respond at glutamate concentrations higher than 15 μMand for only ˜25% of cells (vs. ˜80% in the presence of glycine). Asglycine is an NMDA receptor co-agonist, this behavior further emphasizesthe role these receptors in the glutamate-evoked responses. The maximumamplitude of a typical phase increase is ˜10 degree representing ˜5% ofthe initial cellular phase. If we assume once again a constant cellularrefractive index (n_(s)=1.365), this corresponds to an increase in thevertical (z) axis of ˜0.6 μm.

FIG. 4 a shows an example of the evolution of the OPL averaged over aspecific area of the sample, and gives only a partial idea of the realpotential of the method of the present invention. With DHI, the spatialdistribution of OPL can be defined, with a transverse resolution of forexample 0.3 μm, and OPL time courses, such as presented in FIG. 4 a, canbe obtained for different micro-domains of the cell, or for each pixelof the quantitative phase-contrast image. The recorded signal cantherefore be followed over time and space, for example by constructinganimated sequences, for example by using the processing unit 18.

An informative way to characterize the observed effect consists inmapping the spatial distributions of OPL variations at different times.For the experiment presented in FIG. 4, this representation indicatedthat the observed cell morphology change is anisotropic. Indeed we haveobserved that OPL measurements presented a marked decrease along thecell boundaries, and a global increase over a central region of thesoma, on top of heterogeneous local variations. If we consider anellipse inside the cell body, the observed shape changes consisted in areduction of its semi-minor axis, which enhances the cell eccentricity.Ten minutes after the end of a glutamate pulse application, we observedthat small local OPL variations persist in some areas, even though thecellular OPL has globally recovered its initial level.

The high sensitivity of the present invention has revealed thatglutamate applied during a few seconds at physiological concentrationsinduces reversible morphological changes mediated by NMDA receptors, inmicrodomains of mouse cortical neurons. These results demonstrate thatthe present invention is a new method that offers unique possibilitiesfor real-time 3D finctional imaging with a sub-cellular level of spatialresolution.

Morphological neuronal changes (following pharmacological mediumapplications: Glutamate, MK801.) have been monitored during a few hours.To optimize opto-mechanical stability during such periods of time thewhole holographic microscope may be enclosed in a container. Despitethis enclosure, different noise sources remain (such as coherent noise),extremely sensitive to opto-mechanical instabilities, which may perturbthe reconstructed images. These residual noise sources render thequantitative analysis of neuronal morphology changes very difficult. Toreduce the effects of these residual noise sources we have developed adevice that generates and delivers a controlled energy in the area ofthe biological sample. This controlled energy may be mechanicalvibrations and may be transmitted by perfusion line to the chamber.

The method of the present invention uses in priority a DHI technique tomeasure the phase of a radiation, and in particular the method describedin patent WO 00/20929. However, it is evident that all the conceptsdescribed in the present invention, and in particular the fact that thesample is embedded in a medium, can be extended to other techniques forphase measurements. In particular, the present invention is also relatedto methods using phase measuring techniques such as in-line digitalholography, or Fourier-transform digital holography, or digitalheterodyne holography, or phase-shifting interferometric techniques.Considering that the concept of hologram is a generalization of theconcept of interferogram, or that the concept of hologram is aparticular example of interferogram, any kind of interferometrictechniques can be used in principle, within the context of the presentinvention, as a mean to measure the phase of an electromagnetic wave.

References

Patents

-   U.S. Pat. No. 6,262,218 Cuche et al.-   WO 00/20929 Cuche et al    Other publications-   J. W. Goodmann and R. W. Lawrence “Digital image formation from    electronically detected holograms,” Appl. Phys. Lett, Vol. 11, 1967.    pp.77-79.-   Ichirou Yamaguchi et al. “Image formation in phase-shifting digital    holography and application to microscopy”, Applied Optics, Vol. 40,    No. 34, 2001, pp. 6177-6186.-   W. S. Haddad et al. “Fourier-transform holographic microscope,”    Applied Optics, Vol. 31, 1992, pp. 4973-4978.-   U. Schnars and W. Jüptner, “Direct recording of holograms by a CCD    target and numerical reconstruction,” Applied Optics, Vol. 33, 1994,    pp. 179-181.-   O. Coquoz et al., “Performances of endoscopic holography with a    multicore optical fiber,” Applied Optics, Vol. 34, 1995, pp.    7186-7193.-   E. Cuche et al., “Digital holography for quantitative phase-contrast    imaging”, Optics Letters, Vol. 24, 1999, pp. 291-293.-   E. Cuche et al., “Simultaneous amplitude-contrast and quantitative    phase-contrast microscopy by numerical reconstruction of Fresnel    off-axis holograms”, Applied Optics, Vol. 38, 1999, pp. 6994-7001.-   E. Cuche et al., “Spatial Filtering for Zero-Order and Twin-Image    Elimination in Digital Off-Axis Holography”, Applied Optics, Vol. 38    No. 34, 1999.-   E. Cuche et al., “Aperture apodization using cubic spline    interpolation: Application in digital holographic microscopy”,    Optics Communications, Vol. 182, 2000, pp. 59-69.-   T. Colomb et al., “Polarization Imaging by Use of Digital    Holography”, Applied Optics, Vol. 38, No 34, 1999.-   R. Barer and S. Joseph, in “Refractometry of living cells”,    Quarterly Journal of Microscopical Science, Vol. 95, 1954, pp.    399-423.-   R. Barer in “Refractometry and interferometry of living cells,”    Journal of the Optical Society of America, Vol. 47, 1957, pp.    545-556.-   AJ Coble et al. in “Microscope interferometry of necturus gallblader    epithelium”, Josiah Macy Jr. Fundation, New York, 1982, p. 270-303.-   AC. Svoboda et al in “Direct observation of kinesin stepping by    optical trapping interferometry ”, Nature, Vol. 365, 1993.-   J. Farinas and A. S. Verkman, in “Cell volume plasma membrane    osmotic water permeability in epithelial cell layers measured by    interferometry,” Biophysical Journal, Vol. 71, 1996, pp. 3511-3522.-   G. A. Dunn and D. Zicha in “Dynamics of fibroblast spreading,”    Journal of Cell Science, Vol. 108, 1995, pp. 1239-1249.

1. An apparatus for performing digital holographic imaging (DHI) of asample, said apparatus comprising: an holographic creation unit, anholographic reconstruction unit, a processing unit, a sample unitincluding a container adapted to contain a medium in which a sample iscontained, characterized by the fact that it furthermore comprisesmeasuring means adapted to measure at least one parameter specific ofsaid medium or at least one parameter specific of said sample, saidmedium being different from ambient air.
 2. Apparatus according to claim1 wherein said measuring means are adapted to measure the dielectricproperties of said medium.
 3. Apparatus according to claim 1 whereinsaid measuring means are adapted to measure the index of refraction ofsaid medium.
 4. Apparatus according to claim 1 wherein said measuringmeans are adapted to measure the dielectric properties of said sample.5. Apparatus according to claim 1 wherein said measuring means areadapted to measure the index of refraction of said sample.
 6. Apparatusaccording to claim 1 wherein said measuring means are adapted to measurethe thickness of said sample.
 7. Apparatus according to claim 1 whereinsaid measuring means are adapted to measure the surface topography ofsaid sample.
 8. Apparatus according to claim 1 furthermore comprisingprocessing means adapted to process at least one parameter specific ofsaid medium or one parameter specific of said sample.
 9. Apparatusaccording to claim 8 wherein said processing means are adapted toprocess the dielectric properties of said medium.
 10. Apparatusaccording to claim 8 wherein said processing means are adapted toprocess the index of refraction of said medium.
 11. Apparatus accordingto claim 8 wherein said processing means are adapted to process thedielectric properties of said sample.
 12. Apparatus according to claim 8wherein said processing means are adapted to process the index ofrefraction of said sample.
 13. Apparatus according to claim 8 whereinsaid processing means are adapted to process the thickness of saidsample.
 14. Apparatus according to claim 8 wherein said processing meansare adapted to process the surface topography of said sample. 15.Apparatus according to claim 1 comprising a medium controller unitadapted to modify or to regulate the medium properties, including thepartial or complete medium exchange of a medium in which a sample iscontained.
 16. Apparatus according to claim 1 comprising vibrating meansfor mechanically vibrating at least a part of the sample unit. 17.Apparatus according to claim 16 wherein said vibrating means areincorporated in the medium controller unit.
 18. Method for performingdigital holographic imaging (DHI) of a sample contained in a medium,said method comprising the following steps: producing at least onehologram by interference between at least one reference wave and atleast one object wave, producing at least one digital hologram byacquiring and digitizing at least one hologram, reconstructing at leastone amplitude contrast image and/or at least one quantitative phasecontrast image by numerical reconstruction of at least one digitalhologram, said method being furthermore characterized by the followingstep: processing of at least one amplitude contrast image and/orprocessing of at least one quantitative phase contrast image in order todetermine at least one parameter of the sample and/or in order todetermine at least one parameter of the medium.
 19. Method according toclaim 18 wherein at least one parameter of the medium is modified duringtime and wherein a plurality of holograms are produced and processed inorder to obtain a plurality of amplitude contrast image and/or aplurality of quantitative phase, and wherein said a plurality ofamplitude contrast image and/or said a plurality of quantitative phasecontrast image are processed in order to describe the temporal evolutionof at least one parameter of the sample.
 20. Method according to claim18 wherein at least one parameter of the medium is modified during timeand wherein a plurality of holograms are produced and processed in orderto obtain a plurality of amplitude contrast image and/or a plurality ofquantitative phase, and wherein said a plurality of amplitude contrastimage and/or said a plurality of quantitative phase contrast image areprocessed in order to describe the temporal evolution of at least oneparameter of the medium.
 21. Method according to claim 18 wherein themedium and the sample interact with each other resulting in amodification of at least one parameter of the sample and/or in amodification of at least one parameter of the medium.
 22. Methodaccording to claim 18 wherein the medium is a dispersive medium andwherein the values of the dielectric properties of the medium arechanged by changing the wavelength of the radiation illuminating thesample, and wherein changing the wavelength of the radiationilluminating the sample results in a modification of at least oneparameter of the sample and/or in a modification of at least oneparameter of the medium.
 23. Method according to claim 18 wherein saidparameter of the sample is the thickness of the sample.
 24. Methodaccording to claim 18 wherein said parameter of the sample is thesurface topography of the sample.
 25. Method according to claim 18wherein said parameter of the sample is a dielectric property of thesample.
 26. Method according to claim 18 wherein said parameter of thesample is the index of refraction of the sample.
 27. Method according toclaim 18 wherein said parameter of the medium are the dielectricproperties of the medium.
 28. Method according to claim 18 wherein saidparameter of the medium is the index of refraction of the sample. 29.Method according to claim 18 wherein the dielectric properties of themedium are adjusted in order to increase or to decrease the contrast ofthe amplitude contrast image and/or in order to increase or to decreasethe contrast of the quantitative phase contrast image.